Terminal Deoxynucleotidyl Transferase (TdT) is a unique DNA polymerase which in normal tissues is restricted to cell populations of the thymus and bone marrow. Because of its restricted distribution, it has been used as a marker of T-cell differentiation.
Since the discovery of TdT's association with acute lymphoblastic leukemia, there has been an expandind proliferation of reports on the association of TdT and neoplasms of lymphoid origin. TdT has proved to be a useful marker in classifying acute leukemia, malignant lymphoma and the blast phase of chronic granulocytic leukemia. Its presence provides an objective method of classification, obviating the ambiguities often found when relying upon morphological criteria alone. The importance of proper classification (which is synonomous with `specific diagnosis`) is demonstrated by recent observations of acute undifferentiated leukemia and chronic granulocytic leukemia in blast crisis, where the presence of TdT is predictive of initial responsiveness to vincristine and prednisone.
In addition to functioning as an aid in the proper classification of lymphoid neoplasms, TdT serves as a sensitive monitor of remission and relapse. The enzyme disappears from the blood during remission and reappears up to several months before relapse may be detected by the presence of circulating blasts in the peripheral blood, thereby providing an early warning of the need for revised or repeated chemotherapy.
The conventional method to determine TdT activity involves processing of the sample, while the second stage is the testing of the processed sample for enzymatic activity.
It is a general requirement of enzymology that enzymes originating and functioning in cells must be made accessible to substrates in order that enzymatic activity be detected. As the cell membrane is impermeable to most enzymatic substrates, its integrity must somehow be disrupted. This is accomplished by selectively permeabilizing cells (treatment with cold shock or toluene, DEAE-dextran, or trypsin, or isotonic detergent, or hypotonic shock, etc.), by cell fixation (treatment with organic solvents, or aldehydes, etc.), or by cellular disruption (treatment with detergent in high salt concentration, or sonication). The two methods most commonly used for preparing biological samples for detecting cellular TdT are cellular disruption by sonication or detergent with high salt concentration treatment. Both procedures result in a crude extract of solubililized enzymes, proteins, nucleic acids, DNA, and other crude cellular constituents. If testing for TdT is conducted in a non-cellular media, cellular disruption and enzyme solubilization has already been effected by the process of cell death.
At this point, as a crude extract, TdT can be tested directly; however, a large increase in activity occurs with partial purification. Consequently, in order to maximize sensitivity and minimize sample size, partial purification such as by phosphocellulose choromotogrpahy or DEAE-sephadex may be and preferrably is carried out. Fractions are then tested directly for TdT activity.
The second stage of determining levels of TdT is the testing of the processed sample for enzymatic activity. The assay is performed by incubating the sample with fluorescently or radioactively labeled primers and/or substrates under conditions suitable for the enzymatic production of acid insoluble polydeoxynucleotides which, by reason of either the primer or substrate employed, are labeled for subsequent detection and quantification by known methods.
There are a variety of assay systems in use. The two most common are the cacodylate-Mg.sup.++ system of Bollum and the Tris-Mn.sup.++ system of McCaffrey. The necessary reagents for analysis are the enzyme sample, DNA primer, deoxynucleoside triphosphate substrate, a divalent metal and a buffer solution.
In a first version of the method, wherein a labeled substrate and unlabeled primer are employed, the primer is a single stranded oligodeoxynucleotide, polydeoxynucleotide or activated denatured DNA. In this version of the method, if TdT is present in the sample, incubation of these reagents in the buffer solution at 35.degree. to 37.degree. C. results in the production of unlabeled DNA primers extended with flourescently or radioactively labeled nucleotide substrates to yield an acid insoluble polydeoxynucleotide. The reaction is stopped by addition of cold trichloroacetic acid (TCA) to a concentration of about 5% by weight. The TCA addition precipitates the polydeoxynucleotide formed by the DNA polymerases present in the sample while the unreacted nucleoside triphopsphate substrates remain in the buffer solution. When radioactively labeled substrates are used the polydeoxynucleotides' precipitate is collected on filter paper discs and its radioactively measured by a liquid scintillation counter. Alternatively, when flourescently labeled substrates are employed the polydeoxynucleotide precipitate is collected, centrifuged, washed with cold 5% TCA, redissolved in water and then measured in a fluorometer.
In a second version of the assay method, wherein labeled primers and unlabeled substrates are used, an oligodeoxynucleotide (usually of less than twenty nucleotides for solubility purposes) is used as the primer and incubation results in the production of a polydeoxynucleotide comprising a labeled primer extended by unlabeled substrates. Addition of cold TCA precipitates the polydeoxynucleotide while the oligodeoxynucleotide remains in the buffer solution. The polydeoxynucleotide is collected as described above and its radioactivity or fluorescence, depending upon which type of labeling was used in the primer, is determined.
Many other methods exist for separating the products of reaction from the primers and substrates of the incubation mixture such as ion-exchange, filter discs, thin layer chromotography or dextan-coated charcoal, but the above described procedures are the most commonly employed. The amount of DNA polymerase originally present in the sample is related to the amount of polydeoxynucleotide formed as a function of the sample, primer and substrate amounts used as well as the time period over which such reagents are incubated. The functional relationship is well known.
Present methods for quantifying TdT in biological samples are not specific to TdT. Such methods also respond to the presence of other DNA polymerases contained in the sample and the amount of polydeoxynucleotide formed during incubation is due to the total amount of DNA polymerase present in the sample. Past efforts to prepare the sample extracts in a manner to separate TdT for assay from other common DNA polymerases, such as DNA polymerases .alpha., .beta., and .gamma. normally found in such samples, have only been partially successful. Generally, even when the extract is purified by phosphocellulose chromotagraphy or ion-filtration chromotagraphy, substantial quantities of potentially interfering DNA polymerases .alpha., .beta., and .gamma. co-eluted with TdT. Thus, although partial sample purification by chromatographic methods is desirable to increase TdT activity, maximize assay sensitivity and permits use of minimum sample sizes, it has not provided the answer to quantifying TdT without interference by other DNA polymerases.
Recently, several methods have been developed which purport to be more sensitive and specific to the detection and quantification of TdT in biological samples. See for instance Coleman et al., Cancer Research, Vol. 36, pp. 120-127, January 1976; and Mertelsmann et al., Leukemia Research, Vol. 2, No. 1, pp. 57-69 (1978). The method reported by Coleman employes a cacodylate buffer which is optional for TdT activity while it inhibits the activity of DNA polymerase .alpha. and .beta.. Additionally, when the sample assayed has a sufficiently low enzymatic activity to make the assay results ambiguous, it is reassayed with the addition of 10% ethanol and 10 mM N-ethylmaleimide which acts to completely inhibit TdT activity, thus, distinguishing between the sample activity attributable to DNA polymerase .beta. as opposed to that of TdT. In the method reported by Mertelsmann, enzyme samples are assayed in duplicate, with and without the addition of 100 mM ATP (adenosine triphosphate). ATP specifically inhibits TdT without affecting other cellular DNA polymerases. The difference in enzymatic activity between the duplicate samples is attributable to TdT activity.
Such methods are complicated, time consuming and, by reason of the fact that they measure difference in activity values rather than measuring TdT specifically and directly, are not completely free of ambiguity of results.
More recently, an immunofluorescent method for the detection of TdT has been developed, which provides specificity for TdT. In this method, an antibody is produced in animals immunized to purified TdT. The antibody must also then be purified to avoid non-specific staining. By exposing cells fixed on a microscope slide to the TdT specific antibody, a strong binding occurs between the antibody and immobilized intracellular TdT. Cells with TdT may then be detected in a fluorescent microscope by the method of indirect immunofluorescence. In indirect immunofluorescence the detection of antigen-antibody complexes is accomplished by adding a second, fluorescent tagged antibody that specifically binds to antibodies from the animal used to produce the TdT specific antibody.
The problem with this technique is that even purified antibodies are not homogenous, and their binding affinities will vary from animal to animal, resulting in variable staining and detection of TdT. Also, the production and purification of TdT specific antibodies is technically difficult and expensive.